Breast cancer is the most common form of cancer in women, resulting in approximately 180,000 new cancer cases annually and causing 18% of cancer-related deaths of women in the United States. It is the second leading cause of cancer-related deaths in humans. Despite recent advances in diagnosing and treating breast cancer, the incidence of this disease has steadily risen at a rate of about 1% per year since 1940. It is estimated that a total of 183,400 new patients were diagnosed with breast cancer in 1995, of which 46,240 will die of the disease. Today, the likelihood that a woman living in North America will develop breast cancer during her lifetime is one in eight.
In light of these statistics, efforts to develop new, more effective methods for treating breast cancer have long been of paramount importance in the medical and research communities. Nevertheless, current methods for treating breast cancer remain limited primarily to conventional surgery, radiation treatment and chemotherapy. These treatment methods are frequently insufficient to prevent progression or recurrence of the disease, and are each attended by severe side effects.
In the case of chemotherapy, many compounds have been shown to be effective against cancerous cells. However, the exact mechanisms of action of many chemotherapeutic agents remain unknown, and these agents often incidentally harm or destroy normal cells. In addition, cytocidal and cytostatic agents work best on cancers with large growth indices, i.e., ones whose cells are rapidly dividing. Thus, chemotherapeutic approaches may be less successful against cancers that are pre-metastatic or that are not particularly aggressive in their growth. Moreover, while some chemotherapeutic agents may reduce a tumor mass significantly after one treatment, they may not be amenable to repeated administration to the same patient if the tumor returns, as is usually the case. Some chemotherapeutic agents can only be administered once in a lifetime, while others require several months or years between treatments. Finally, most become ineffective due to the development of multi-drug resistance by the tumors.
In view of these drawbacks associated with conventional chemotherapy, greater attention in the medical and research communities has been drawn toward developing hormonal therapy agents for treating or preventing breast cancer. One promising class of agents in this context, which has now become a widely used and effective tool against breast cancer, is the anti-estrogen compounds tamoxifen and raloxifene. Tamoxifen and raloxifene belong to a class of pharmaceutical agents termed selective estrogen receptor inhibitors (SERIs). About two thirds of breast tumors express the estrogen receptor-α (ER). Many of these ER positive tumors appear dependent on estrogen for growth and survival, and thus may respond to treatment with anti-estrogens. Tamoxifen has proven to be a successful treatment agent in this context. Unfortunately, the remaining one third of breast cancers which are ER-negative at the time of diagnosis generally do not respond to endocrine therapy. In addition, acquired resistance to tamoxifen in ER-positive tumors is common. For these patients, there is clearly a need for new and better treatment options.
Ideally, new therapeutic and prophylactic agents against breast cancer will target important biological pathways in breast cell growth and differentiation. With respect to developing new hormonal treatment strategies, a large number and variety of hormones and growth factors are thought to interact in complex pathways to influence breast cancer initiation and disease progression. Examples of hormonal regulatory factors that may be involved in such interactions include somatostatin, mammostatin, vasopressin, mammary-derived growth inhibitor (MDGI), mammary-derived growth factor-1 (MDGF-1), inhibins, activins, androgens, glucocorticoids, vitamin D, thyroid hormones, ecosinoids, and oxytocin. However, the contributions of these diverse hormones and growth factors to the initiation and progression of breast cancer remain poorly understood. Even the relatively well known effects of estrogens and anti-estrogens on breast cells appear to depend on interactions among a variety of agents and pathways. These interactions may vary significantly among individual breast tumors, for example depending on genetic or environmental variables such as oncogene activation or the presence or absence of tumor suppressors. Accordingly, a better understanding of how cancer cells circumvent their dependency on normal growth and developmental signals and pathways is of paramount interest.
Among the many hormonal regulatory factors that have been investigated as possible tools for regulating breast cell growth, differentiation and/or survival, the peptide hormone oxytocin has received recent interest as a potential growth modulating agent for breast cancer cells. Human breast cancer cell lines and biopsy samples have been reported to express the oxytocin receptor (OR), as have normal breast myoepithelial and epithelial cells and intraductal cells in benign hyperplastic lesions (Taylor et al., Cancer Res. 50:7882-7886, 1990; Cassoni et al., Virchows Archiv. 425:467-472, 1994; Bussolati et al., Am. J. Pathol. 148:1895-1903, 1995; Planchon et al., Mol. Cell. Endocrinol. 111:219-223, 1995; Ito et al., Endocrinology 137:773-779, 1996; Kimura et al., Human Reprod. 13:2645-2653, 1998; Sapino et al., Anticancer Res. 18:2181-2186, 1998). Several of these and related reports suggest that oxytocin can modulate growth and/or differentiation of breast cancer cells (Taylor et al., Cancer Res. 50:7882-7886, 1990; Cassoni et al., Virchows Archiv. 425:467-472, 1994; Cassoni et al., Int. J. Cancer 66:817-820, 1996; Cassoni et al., Int. J. Cancer 72:340-344, 1997; Sapino et al., Anticancer Res. 18:2181-2186, 1998).
In one study, oxytocin was reported to inhibit proliferation of undifferentiated stem cells in the mouse mammary gland, while increasing the relative number of differentiated myoepithelial and epithelial cells (Sapino et al., Endocrinology 133:838-842, 1993). In another study, the effects of oxytocin and an oxytocin analog, F314, were investigated on cell cultures and xenographs of mouse mammary and colon carcinomas and rat mammary carcinoma (Cassoni et al., Int. J. Cancer 66:817-820, 1996). Both cell proliferation and tumor growth were reportedly inhibited by oxytocin and F314. Additional reports by the same research group concluded that oxytocin inhibits proliferation of human bormone-dependent MCF7 and hormone-independent MDA-MB231 breast cancer cells in vitro and enhances the known inhibitory effect of tamoxifen on estrogen-dependent MCF7 cells and TS/A (Cassoni et al., Virchows Archiv. 425:467-472, 1994; Cassoni et al., Int J. Cancer 66:817-820, 1996; Cassoni et al., Int. J. Cancer 72:340-344, 1997; Sapino et al., Anticancer Res. 18:2181-86, 1998). Based on the accumulated data from these reports, the authors propose that oxytocin may mediate a spectrum of different cellular responses, in different signal-transduction systems, in cells with different phenotypes, and in combination with other mammotrophic hormones through yet undefined mechanisms and pathways.
In view of these reports, there remains a great deal of uncertainty concerning the possible effects of oxytocin and other hormonal regulatory factors on breast cell growth, differentiation and survival. This uncertainty is underscored by a number of conflicting reports about the nature and activity of oxytocin as a regulatory factor in breast cell development. For example, Taylor et al., Cancer Res. 50:7882-7886, 1990, report that oxytocin is mitogenic for estrogen-dependent MCF7 cells—an opposite conclusion to that rendered by the Cassoni research group in the series of reports discussed above. The mitogenic activity of oxytocin observed by Taylor and coworkers was shared by another peptide hormone, vasopressin. However, vasopressin was observed to be mitogenic for MCF7 cells only at low doses, and to exert an opposite, anti-proliferative effect on these same cells at higher doses. In a separate report Sapino et al. (Anticancer Res. 18:2181-86, 1998) state that oxytocin exerts an independent “trophic effect” on breast myoepithelial cells that induces their proliferation and differentiation. In yet another conflicting study, Ito and coworkers (Endocrinology 137:773-779, 1996) report that “the effects of OT (oxytocin) on the growth of cultures breast cancer cells are inconsistent in the short term”, and that available data suggest “that OT does not influence the morphological differentiation of the cancer cell. These collective reports provide insufficient insight and guidance regarding the potential utility of oxytocin, oxytocin analogs, and other hormonal factors as therapeutic agents for successful prophylaxis and treatment of breast cancer.
Whereas the role of oxytocin in breast cell development remains largely undefined, this peptide hormone has well characterized activities for stimulating milk let-down and inducing uterine contraction in mammalian subjects (see, e.g., Boucher et al., J. Perinatology 18:202-207, 1998; Cort et al., Am. J. Vet. Res. 43:1283-1285, 1982). In the clinical setting, oxytocin is routinely used as a labor-inducing agent and during postpartum care or cesarean section to prevent uterine atony and to control bleeding or hemorrhage after delivery of the placenta. It is also widely used as a treatment agent to enhance milk letdown in lactating patients, which activity involves stimulation of contraction by myoepithelial cells surrounding the mammary alveoli. Because oxytocin has a relatively short half-life of only about 4 to 10 minutes in the human system, it must generally be administered by continuous intravenous (IV) infusion to achieve desired uterotonic and milk let-down effects (Boucher et al., J. Perinatology 18:202-207, 1998). However, long-term, repeated or high dose administrations of oxytocin may be attended by substantial side effects.
Oxytocin has also been implicated as a potential factor in certain psychiatric disorders. For example, based on a review of evidence from animal studies demonstrating that the nonapeptides, oxytocin and vasopressin, have unique effects on the normal expression of species-typical social behavior, communication and rituals, Insel and colleagues have proposed that oxytocin or vasopressin neurotransmission may account for several features associated with autism. (Inset et al., Biol. Psychiatry 45:145-157, 1999). A study on autistic children reported that such children had significantly lower levels of plasma oxytocin than normal children. Elevated oxytocin levels were associated with higher scores on social and developmental tests in non-autistic children, but associated with lower scores in autistic children, suggesting that altered oxytocin levels may be associated with autism in children (Modahl et al., Biol. Psychiatric 43:270-277, 1998). A role for oxytocin in obsessive compulsive disorders has also been proposed (Leckman et al., Psychoneuroendocrinology 19:723-749, 1994; but see Altemus et al., Biol. Psychiatry 45:931-33, 1999). In particular, elevated levels of oxytocin have been proposed to affect certain obsessive-compulsive behaviors, such as excessive worrying, sexual compulsions and/or compulsive washing and cleaning. (Leckman et al., Psychoneuroendocrinology 19:723-749, 1994; Leckman et al., Arch Gen Psychiatry 51:782-92, 1994). Elevated levels of oxytocin have also been implicated in Prader-Willi syndrome, a genetic disorder associated with mental retardation, appetite dysregulation and a risk of developing obsessive compulsive disorder (Martin et al., Biol. Psychiatric 44:1349-1352, 1998). One study found that intranasal administration of oxytocin was not effective, however, as an anticompulsive agent (den Boer and Westenberg, Peptides 13:1083-85, 1992).
A number of oxytocin analogs have been evaluated as possible substitute agents for inducing uterine contraction and milk let-down in mammalian patients with the goal of minimizing oxytocin's side effects. One such analog, carbetocin (1-butanoic acid-2-(O-methyl-L-tyrosine)-1-carbaoxytocin, or, alternatively, deamino-1 monocarba-(2-O-methyltyrosine)-oxytocin [d(COMOT)]) is a long-acting synthetic oxytocin analog which exhibits both utcrotonic and milk let-down inducing activities (Atke et al., Acta Endocrinol. 115:155-160, 1987; Norstrom et al., Acta Endocrinol. 122:566-568, 1990; Hunter et al., Clin. Pharmacol. Ther. 52:60-67, 1992; Silcox et al., Obstet Gynecol. 82:456-459, 1993; Vilhardt et al., Pharmacol. Toxicol. 81:147-150, 1997; Boucher et al., J. Perinatology 18:202-207, 1998). The half-life of carbetocin is reportedly 4 to 10 times longer than that of oxytocin, which is reflected in substantial prolongation of the uterotonic and milk let-down inducing activities of this analog. This apparent increase in metabolic stability is attributed to N-terminal desamination and replacement of a 1-6 disulfide bridge by a methylene group in carbetocin, which modifications are thought to protect this analog from aminopeptidase and disulfidase cleavage (Hunter et al., Clin. Pharmacol. Ther. 52:60-67, 1992).
Despite these apparent advantages of carbetocin over its parent molecule oxytocin, it is widely noted that modifications of peptides and proteins can substantially reduce or even abolish biological activities in the modified analog (see, e.g., Vilhardt et al., Pharmacol. Toxicol. 81:147-150, 1997). This appears to be at least partially the case for carbetocin, based on reports that the potency of this analog is reduced in vivo to as little as one-tenth the potency of native oxytocin (Hunter et al., Clin. Pharmacol. Ther. 52:60-67, 1992). Another potential drawback to using peptide analogs such as carbetocin is that, in addition to having diminished potency, they may also act as antagonists to inhibit activities of their native counterparts (e.g., by competitive binding with a target receptor). In this context, reports suggest that carbetocin, while exhibiting some degree of agonist activity, also acts as an antagonist against native oxytocin (Engstrom et al., Eur. J. Pharmacol. 355:203-210, 1998). Yet another concern for using peptide analogs relates to their potential side effects. In the case of carbetocin, dose acceleration studies have revealed significant toxicity of this analog in clinical settings (van Dongen, Eur. J. Obstet. Ganecol. Reprod. Biol. 77:181-187, 1998). Among 45 women who received between 15 μg-200 μg of carbetocin by intramuscular injection within 24 hours of childbirth, seven women suffered serious adverse side effects. Six cases presented with blood loss of at least 1000 ml. Four cases required manual placenta removal. Five cases required additional oxytocics administration and five required blood transfusion.
In view of the foregoing, there remains an urgent need in the art for novel tools and methods to manage and treat breast cancer, psychiatric disorders and other conditions in which abnormal oxytocin levels are implicated. In particular, new biological targets must be ascertained and novel therapies designed to manage and treat breast cancers that are not subject to treatment by conventional chemotherapeutic methods or anti-estrogen therapies. Such new agents could be used alone or in combination with chemotherapy or anti-estrogen treatment to improve patient outcomes. Similarly, new biological agents must be developed to manage and treat psychiatric disorders that are not subject to treatment by conventional therapies. Such new agents could be used alone or in combination with existing drug regimens to improve patient outcomes. Surprisingly, the methods and compositions of the present invention fulfill these needs and satisfy other objects and advantages that will become apparent from the description which follows.